Biology subjects

Tirian, L.

Publications and source records attributed to Tirian, L..

7 recordsLinked to original sources

bioRxiv2025

RNA decay via the nuclear exosome is essential for piwi-mediated transposon silencing

Nuclear Argonaute proteins safeguard genome integrity by directing transcriptional silencing and heterochromatin formation at transposon loci. Yet it remains unclear how Argonautes enforce robust repression while relying on target transcription for their own recruitment. Here we show that transposon silencing by the Drosophila nuclear Piwi-piRNA pathway requires degradation of target RNA by the nuclear exosome. Using proximity proteomics at endogenous Piwi target sites, we identify two previously uncharacterized paralogs, TEsup-1 and TEsup-2, as essential cofactors for Piwi-mediated silencing. TEsup proteins act in part by engaging nuclear exosome adaptor complexes at piRNA-targeted transcripts through a domain that recognizes proline-rich peptides. Disruption of the Piwi-TEsup-exosome axis leads to accumulation and nuclear export of piRNA-targeted transposon RNAs. Notably, the P-element--which evades heterochromatin-based repression--is silenced primarily through this RNA-decay pathway. Thus, the nuclear piRNA pathway couples target recognition to RNA degradation, reconciling small RNA-guided heterochromatin formation with ongoing transcription at target loci.

molecular biology↗

bioRxiv2025

Molecular basis of polyadenylated RNA fate determination in the nucleus

Eukaryotic genomes generate a plethora of polyadenylated (pA+) RNAs1,2, that are packaged into ribonucleoprotein particles (RNPs). To ensure faithful gene expression, functional pA+ RNPs, including protein-coding RNPs, are exported to the cytoplasm, while transcripts within non-functional pA+ RNPs are degraded in the nucleus1-4. How cells distinguish these opposing fates remains unknown. The DExD-box ATPase UAP56/DDX39B is a central component of functional pA+ RNPs, promoting their docking to the nuclear pore complex (NPC)-anchored transcription and export complex 2 (TREX-2) (ref.5,6), which triggers transcript release from UAP56 to facilitate export (ref.7,8). Here, we uncover that the Poly(A) tail exosome targeting (PAXT) connection9 harbors its own TREX-2-like module, which releases pA+ RNAs from UAP56 for decay by the nuclear exosome. The core of this module consists of a LENG8-PCID2-SEM1 (LENG8-PS) trimer, which we show is structurally and functionally equivalent to the central GANP-PCID2-SEM1 (GANP-PS) trimer of TREX-2. Mutagenesis and transcriptomic data demonstrate that the nuclear fate of pA+ RNPs is governed by the contending actions of nucleoplasmic PAXT and NPC-associated TREX-2, which interpret RNA-bound UAP56 as a signal for RNA decay or export, respectively. As RNA targets of PAXT are generally short and intron-poor, we propose an overall model for pA+ RNP fate determination, whereby the distinct sub-nuclear localizations of PAXT and TREX-2 govern the degradation of short non-functional pA+ RNAs while allowing export of their longer and functional counterparts.

molecular biology↗

bioRxiv2025

In vivo crosslinking and effective 2D enrichment for interactome studies of the nucleosome.

Cross-linking mass spectrometry has evolved as a powerful technique to study protein-protein interactions and to provide structural information over the past decades. Low reaction efficiencies, and complex matrices lead to challenging system wide crosslink analysis. In this study, we improved and streamlined an Azide-A-DSBSO based in vivo crosslinking workflow employing two orthogonal effective enrichment steps: Affinity enrichment and size exclusion chromatography (SEC). Combined, they allow an effective pulling of DSBSO containing peptides and remove the background of linear as well as mono-linked peptides. We found that the analysis of a single SEC fraction is effective to yield [~]90% of all crosslinks, which is important whenever measurement time is limited, and sample throughput is crucial. Our workflow resulted in more than 5000 crosslinks from K562 cells and generated a comprehensive PPI network from whole cells as well as nuclear extracts. From 393 PPI found within the nucleus, 56 have not yet been reported in the STING database and are representing a valuable resource for investigating new molecular mechanisms and provide complementary data for future studies. We further show, that by applying DSBSO to nuclear extracts we yield more crosslinks on lower abundant proteins and showcase this on the DEAD-box RNA helicase DDX39B which is predominantly expressed in the nucleus. Our data indicates that DDX39B is present in monomeric and dimeric form together with DDX39A within the nuclear extracts analyzed.

biochemistry↗

bioRxiv2024

A molecular switch orchestrates the nuclear export of human messenger RNA

The nuclear export of messenger RNA (mRNA) is a key step in eukaryotic gene expression (Kohler and Hurt, 2007). Despite recent insights into the packaging of newly transcribed mRNAs into ribonucleoprotein complexes (mRNPs) (Pacheco-Fiallos et al., 2023; Bonneau et al., 2023), the subsequent events that govern mRNA export are poorly understood. Here, we elucidate the molecular basis of human mRNA export licensing, which involves the remodeling of mRNP-bound transcription-export complexes (TREX), the formation of export-competent mRNPs, the docking of mRNPs at the nuclear pore complex (NPC), and the release of mRNPs at the NPC to initiate export. Our biochemical and structural data uncover the ATPase DDX39/UAP56 as a central molecular switch that directs mRNPs through the TREX and the NPC-anchored TREX-2 complexes using its ATPase and mRNA-binding cycle. Collectively, these findings establish a mechanistic framework for a general and conserved mRNA export pathway.

molecular biology↗

bioRxiv2021

A SUMO-dependent regulatory switch connects the piRNA pathway to the heterochromatin machinery in Drosophila

Nuclear Argonaute proteins, guided by small RNAs, mediate sequence-specific heterochromatin formation. The molecular principles that link Argonaute-small RNA complexes to cellular heterochromatin effectors upon binding to nascent target RNAs are poorly understood. Here, we elucidate the mechanism by which the PIWI interacting RNA (piRNA) pathway connects to the heterochromatin machinery in Drosophila. Piwi-mediated stabilization of the corepressor complex SFiNX on chromatin leads to SUMOylation of its subunit Panoramix. SUMOylation, together with an amphipathic LxxLL motif in Panoramixs intrinsically disordered repressor domain, are necessary and sufficient to recruit small ovary (Sov), a multi-zinc finger protein essential for general heterochromatin formation and viability. Structure-guided mutations that abrogate the Panoramix-Sov interaction or that prevent SUMOylation of Panoramix uncouple Sov from the piRNA pathway, resulting in viable but sterile flies in which Piwi-targeted transposons are derepressed. Thus, by coupling recruitment of a corepressor to nascent transcripts with its SUMOylation, Piwi engages the heterochromatin machinery specifically at transposon loci.

molecular biology↗

bioRxiv2019

A heterochromatin-specific RNA export pathway facilitates piRNA production

PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30nt piRNAs are processed in the cytoplasm from long non-coding RNAs. How piRNA precursors, which often lack RNA processing hallmarks of export-competent transcripts, achieve nuclear export is unknown. Here, we uncover the RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1, and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. Our findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to achieve export of heterochromatic, unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.

molecular biology↗

bioRxiv2017

The VT GAL4, LexA, and split-GAL4 driver line collections for targeted expression in the Drosophila nervous system

In studying the cellular interactions within complex tissues, it is extremely valuable to be able to reproducibly and flexibly target transgene expression to restricted subsets of cells. This approach is particularly valuable in studying the nervous system, with its bewildering diversity of neuronal cell types. We report here the generation of over 18,000 driver lines (the VT collection) that exploit the GAL4, LexA, and split-GAL4 systems to express transgenes in distinct and highly specific cell types in Drosophila. We document the expression patterns of over 14,000 of these lines in the adult male brain.

neuroscience↗